Abstract NLGNs are known to play crucial roles in synaptogenesis and the maturation of the postsynaptic density through recruitment of and interactions with scaffolding proteins, neurotransmitter receptors, and ion channels. It has been shown that NRXN binding is crucial for NLGN function, as NRXN is capable of clustering NLGNs at the postsynaptic density (PSD) and initiating recruitment of downstream interacting proteins necessary for synapse stabilization and maturation. Most vertebrates possess four NLGN genes: NLGN1, NLGN2, NLGN3, and NLGN4. In most mammals, NLGN4 is located on the X chromosome, however in higher order primates such as Homo sapiens and Pan troglodytes NLGN4X is complemented on the Y chromosome (sometimes referred to as NLGN5) with nearly 98% homology to NLGN4X. It is believed that this NLGN4Y isoform originated from a duplication event that occurred relatively recently during evolution Unlike almost all vertebrates, humans possess X chromosome and Y chromosome isoforms of NLGN4, however the function of NLGN4 at synapses remains unknown. We intend to investigate the functional properties of NLGN4X and NLGN4Y such as NRXN binding, synaptogenesis, and localization. We plan on addressing these questions with the following three specific aims: 1) Determine whether NLGN4X and NLGN4Y are localized to human synaptic sites. 2) Determine the affinities of NLGN4X and NLGN4Y for NRXN. 3) Determine the molecular mechanism responsible for differences in the synaptogenic activity of NLGN4X and NLGN4Y. We have conducted experiments to demonstrate that we can achieve the goals of each of the three ambitious aims we proposed. For specific aim 1, we established approaches to study NLGN function in human brain tissue and cells. For Specific aim 2, we have established an assay and begun experiments to determine the affinity of NLGN4 in biological sex-specific combinations. For specific aim 3, we have begun experiments using a synapse assay system and expressed NLGN4X and NLGN4Y and generated constructs to begin structure function analysis. Together these preliminary data indicate that we are poised to make rapid progress on our understanding of these sexually dimorphic genes. Results are likely to shed light on the matter of synaptic dysfunction in disorders such as ASD and learning disabilities and propose a potential difference in synaptic function between males and females.